连作地黄cDNA消减文库的构建及分析

Construction and analysis of suppression subtractive cDNA libraries of continuous monoculture Rehmannia glutinosa

目的:通过构建连作地黄cDNA消减文库,探讨地黄连作障碍的分子机制。方法:利用抑制性消减杂交(SSH)技术构建连作地黄的正反消减文库,通过蓝白斑筛选、PCR的方法鉴定出阳性克隆,并对其进行测序和生物信息学分析。结果:连作地黄cDNA消减文库构建成功,正向和反向消减文库均筛选了300个阳性克隆。测序结果表明:正库、反库分别获得232条、214条特异的EST序列;经NCB I数据库分析,正库、反库中分别有200,195条EST序列的基因具有蛋白功能注释;COG基因功能预测结果表明,正库、反库中分别有60,61条EST序列具有相应的的基因功能分类,涉及21个代谢途径。结论:差异表达基因的功能注释表明,连作对地黄体内的基因表达具有深刻的影响。本研究筛选地黄响应连作的关键基因,为揭示地黄连作障碍的分子机制奠定了基础。

Objective： To explore the molecular mechanism of continuous monoculture problem by constructing the cDNA libraries of continuous monocuhure Rehmannia glutinosa. Method： To use the suppression subtractive hybridization （SSH） technique to construct the forward and reverse subtractive cDNA libraries of continuous monocuhure R. glutir^osa to adopt blue-white colony screen- ing and PCR to detect the positive clones which would be sequenced and analyzed by bioinformatics. Result： The subtracted cDNA li- braries of continuous monoculture R. glutinosa, were successfully constructed, and the result showed that the forward and reverse sub- tracted libraries obtained 300 positive clones, respectively. The forward and reverse libraries got different ESTs, and produced 232 （forward library） and 214 （reverse library） unique ESTs by sequencing. Based on homology search of BLASTX and BLASTN in NC- BI, 200 and 195 of unique ESTs were homologous to known genes in the forward and reverse libraries, respectively. Categories of or- thologous group （COG） showed that the forward and reverse libraries got 60 and 61 ESTs with the corresponding gene annotation, in- volving 21 metabolic pathways. Conclusion： The information of differential expression genes in continuous monocuhure R. glutinosa, and their functional annotation of differentially expressed genes indicate that continuous monocuhure has a profound effect on expression of the genes in R. glutinosa. Furthermore, the research analyzed several key genes in response to replant problem, which provided a foundation for revealing the molecular mechanism of continuous monocuhure R. glutinosa.